Provided are methods for preparing samples for sequencing. Also provided are methods for sequence analysis. Also provided are methods for classifying multiple aspects of a nucleotide repeat expansion disorder in a single sequencing assay. Also provided are methods for genotyping a target nucleic acid sequence.

The methods, compositions, and kits of the disclosure provide a novel approach for a whole genome, unbiased DNA analysis method that can be performed on limited amounts of DNA. can be used to analyze DNA to determine its modification status. Aspects of the disclosure relate to a method for amplifying bisulfite-treated deoxyribonucleic acid (DNA) molecules comprising: (a) ligating an adaptor to the DNA molecules, wherein the adaptor comprises a RNA polymerase promoter comprising bisulfite-protected cytosines; (b) treating the ligated DNA molecules with bisulfite; (c) hybridizing the bisulfite-treated DNA molecules with a primer; (d) extending the hybridized primer to make double stranded DNA; and (e) in vitro transcribing the double-stranded DNA to make RNA.

The methods, compositions, and kits of the disclosure provide a novel approach for a whole genome, unbiased DNA analysis method that can be performed on limited amounts of DNA. can be used to analyze DNA to determine its modification status. Aspects of the disclosure relate to a method for amplifying bisulfite-treated deoxyribonucleic acid (DNA) molecules comprising: (a) ligating an adaptor to the DNA molecules, wherein the adaptor comprises a RNA polymerase promoter comprising bisulfite-protected cytosines; (b) treating the ligated DNA molecules with bisulfite; (c) hybridizing the bisulfite-treated DNA molecules with a primer; (d) extending the hybridized primer to make double stranded DNA; and (e) in vitro transcribing the double-stranded DNA to make RNA.

Nuclease activity can affect the methylation level and fragmentation of cfDNA. Certain levels of nuclease activity may be correlated with certain levels of methylation in certain regions. Methylation level in certain genomic regions can be analyzed to classify nuclease activity. Methylation statuses of different genomic regions compared to methylation statuses of other genomic regions can determine a level of a condition (e.g., a disease such as cancer or disorder) in a subject. Nuclease activity can be monitored through analysis of methylation statuses of different sites. The efficacy of a treatment can also be determined using methylation levels at certain genomic regions. The number of fragments from genomic regions that are hypomethylated or hypermethylated in a reference genome can be used to provide information (e.g., fractional concentration) on the sample itself. The size distribution of extrachromosomal circular DNA can also be used to analyze a biological sample. Systems are also described.

Nuclease activity can affect the methylation level and fragmentation of cfDNA. Certain levels of nuclease activity may be correlated with certain levels of methylation in certain regions. Methylation level in certain genomic regions can be analyzed to classify nuclease activity. Methylation statuses of different genomic regions compared to methylation statuses of other genomic regions can determine a level of a condition (e.g., a disease such as cancer or disorder) in a subject. Nuclease activity can be monitored through analysis of methylation statuses of different sites. The efficacy of a treatment can also be determined using methylation levels at certain genomic regions. The number of fragments from genomic regions that are hypomethylated or hypermethylated in a reference genome can be used to provide information (e.g., fractional concentration) on the sample itself. The size distribution of extrachromosomal circular DNA can also be used to analyze a biological sample. Systems are also described.

The present disclosure provides a method for methylation analysis of genomic fragments.

The present disclosure provides a method for methylation analysis of genomic fragments.

The present invention relates to a method to determine bisulfite conversion of unmethylated cytosine to uracil in genomic DNA, comprising the steps of providing a first set of amplification primers for amplifying bisulfite converted copies of a repetitive DNA element by qPCR and a second set of amplification primers for amplifying unconverted copies of said repetitive DNA element by qPCR, performing a multiplex qPCR with said first and second set of amplification primers to generate amplicons, and determining the bisulfite conversion efficiency by comparing the amounts of said first and second amplicon.

The present invention relates to a method to determine bisulfite conversion of unmethylated cytosine to uracil in genomic DNA, comprising the steps of providing a first set of amplification primers for amplifying bisulfite converted copies of a repetitive DNA element by qPCR and a second set of amplification primers for amplifying unconverted copies of said repetitive DNA element by qPCR, performing a multiplex qPCR with said first and second set of amplification primers to generate amplicons, and determining the bisulfite conversion efficiency by comparing the amounts of said first and second amplicon.

The present invention provides a method for detecting a target nucleic acid that discriminates the target nucleic acid from a non-target nucleic acid having a nucleotide sequence or modification state that differs from a portion of the target nucleic acid, the method comprising conducting a nucleic acid amplification reaction using a region in the non-target nucleic acid that differs from the target nucleic acid as a target region, using a region in the target nucleic acid that differs from the non-target nucleic acid as a corresponding target region, using a nucleic acid test sample as a template, and using a primer that hybridizes with both the target nucleic acid and the non-target nucleic acid, with the nucleic acid amplification reaction conducted in the presence of a molecule capable of binding specifically to the target region in the non-target nucleic acid, under temperature conditions under which the molecule can bind to the non-target nucleic acid, and then detecting the target nucleic acid on the basis of the presence or absence of an amplification product.